Human monoclonal antibodies derived from human B cells and having neutralizing activity against influenza A viruses

ABSTRACT

The present invention relates to human monoclonal antibodies derived from human B cells present in the blood of patients who had recovered from infection with influenza A viruses, wherein the monoclonal antibodies have neutralizing activity against influenza A viruses. The anti-influenza A virus monoclonal antibody of the present invention has binding and neutralizing activities against at least one influenza A virus selected from the group consisting of influenza A virus H1, H2 and H5 subtypes, and thus it is useful for the prevention and treatment of a disease caused by the influenza A virus and is also useful for diagnosis of influenza A virus infection.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 13/583,529, filed on Sep. 7, 2012, which is a U.S. national phaseapplication, pursuant to 35 U.S.C. §371, of PCT/KR2011/001563, filedMar. 7, 2011, designating the United States, which claims priority toKorean Application No. 10-2010-0020587, filed Mar. 8, 2010. The entirecontents of the aforementioned patent applications are incorporatedherein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The contents of the text file named 48169-569C01US_ST25.TXT, which wascreated on Feb. 2, 2017, and is 35,635 bytes in size, are herebyincorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to human monoclonal antibodies derivedfrom human B cells present in the blood of patients who had recoveredfrom infection with influenza A viruses, wherein the monoclonalantibodies have neutralizing activity against influenza A viruses.

BACKGROUND ART

Influenza, an illness caused by respiratory infection with influenzaviruses, often occurs in winter. It is known to have very highinfectivity and to affect all age groups, particularly elderly people(Treanor J, 2004, N Etzgl J Med. 350(3):218-20). Influenza virus is anegative-stranded and enveloped RNA (ribonucleic acid) virus belongingto the family Orthomyxoviridae. This family has eight segments ofsingle-stranded RNA and are classified as influenza types A, B and C.Influenza A viruses are further divided into subtypes on the basis oftheir major surface proteins hemagglutinin (HA) and neuraminidase (NA).Up to date, 16 HAs and 9 NAs have been identified (Cheung T K and Poon LL 2007, Ann NY Acad Sci. 1102:1-25). Influenza viruses infect a widerange of animals including birds, pigs and humans depending on theirtypes and have a genome composed of segmented RNAs. For this reason,influenza viruses can continuously mutate and recombine, resulting innew genetic variations (Treanor J, 2004. N Engl J Med. 350(3):218-20).For this reason, it is difficult to obtain permanent immunity againstinfluenza viruses. The most effective prevention method currently usedis vaccination against particular influenza viruses expected to beprevalent.

Influenza Vaccines are generally produced using eggs, but this is aninefficient method that requires much time. Accordingly, this method hasa problem in that it is difficult to produce sufficient amounts ofvaccines each year within a limited time frame. To solve this problem,studies on methods of producing vaccines by cell culture are beingactively conducted in several pharmaceutical companies (GSK, Baxter,etc.). In addition, it is very difficult to develop a vaccine rapidlyagainst the pandemic influenza virus when pandemic infection occurs.Also, antiviral drugs are not completely reliable due to a problemassociated with the appearance of mutant viruses having resistance.

To solve this problem, recently antibodies against influenza viruseshave been actively developed for a therapeutic purpose (Throsby et al,2008, PloS One 3 (e3942); Sui et al., 2009, Nature structural &molecular biology. 16 (265-273); Simmons et al, 2007, PloS Medicine 4(e178)).

Blood products from recovered patients have been used to treat patientsinfected with various viruses, as well as to treat pandemic fluinfections. For example, when patients infected with Spanish influenzavirus had symptoms of pneumonia, blood products collected from patientswho recovered from infection with the flu are used to treat the flu(Luke et al., 2006. Annals of internal medicine. 145:599). As such,hyperimmune globulin (IgIv) is purified from human plasma and used totreat patients infected with various viruses, but the product obtainedas described above may not be safe from potential infectious agents inblood and is inefficient for mass production.

Human B cells are used for the screening of specific human monoclonalantibodies. However, immortalization of human B cells by Epstein-Barrvirus (EBV) is inefficient in immortalization of B-cells and istime-consuming. To overcome this inefficiency, new techniques are beingdeveloped and used. One of these techniques is to use an RTPCR method toobtain genetic information for an antibody directly from B cells. Forexample, there is a method comprising staining B cells that express anantibody to a specific antigen, isolating the B cells using a FACSsorter, obtaining genetic information for the antibody from the single Bcells by an RT-PCR method, inserting the genetic information into anexpression vector, and transfecting the expression vector into animalcells, thereby producing a large amount of the antibody. To perform sucha production in an easier and rapid manner, the following technique canbe used. The new technique “immunospot array assay on a chip” (ISAAC)enables an antibody gene to be obtained by screening single B cells,which secrete a specific monoclonal antibody, within several weeks (Jinet al., 2009 Nat Med. 15, 1088-1092). The antibody thus obtained is anatural human antibody which can be more effective in terms ofimmunogenic issues.

SUMMARY

It is an object of the present invention to provide a human monoclonalantibody, which is derived from human B cells and has neutralizingactivity against influenza A virus.

Another object of the present invention is to provide an isolatednucleic acid molecule encoding said monoclonal antibody.

Still another object of the present invention is to provide anexpression vector containing said nucleic acid molecule insertedtherein.

Still another object of the present invention is to provide anantibody-producing cell line transfected with said expression vector.

Still another object of the present invention is to provide a method forscreening a human monoclonal antibody.

Still another object of the present invention is to provide acomposition comprising said human monoclonal antibody.

Still another object of the present invention is to provide a method oftreating a disease caused by influenza A virus using said humanmonoclonal antibody.

Still another object of the present invention is to provide a method ofpreventing a disease caused by influenza A virus using said humanmonoclonal antibody.

Still another object of the present invention is to provide a method fordiagnosis of influenza A virus infection using said human monoclonalantibody.

Yet another object of the present invention is to provide a kit fordiagnosis of influenza A virus, which comprises said human monoclonalantibody.

To achieve the abovegoals, the present invention provides ananti-influenza A virus monoclonal antibody having neutralizing activityagainst at least one influenza A virus selected from the groupconsisting of influenza A virus H1, H2 and H5 subtypes.

The present invention also provides an isolated nucleic acid moleculeencoding said monoclonal antibody.

The present invention also provides an expression vector containing saidisolated nucleic acid molecule inserted therein.

The present invention also provides an antibody-producing cell linetransfected with said expression vector.

The present invention also provides a method for screening a humanmonoclonal antibody.

The present invention also provides a composition comprising said humanmonoclonal antibody.

The present invention also provides a composition for preventing andtreating a disease caused by influenza A virus, the compositioncomprising said human monoclonal antibody.

The present invention also provides a composition for diagnosis ofinfluenza A virus infection, the composition comprising said humanmonoclonal antibody.

The present invention also provides a method of treating a diseasecaused by influenza A virus using said human monoclonal antibody.

The present invention also provides a method of preventing a diseasecaused by influenza A virus using said human monoclonal antibody.

The present invention also provides a method for diagnosis of influenzaA virus infection using said human monoclonal antibody.

The present invention also provides a kit for diagnosis of influenza Avirus, which comprises said human monoclonal antibody.

The anti-influenza A virus monoclonal antibody of the present inventionhas binding and neutralizing activities against at least one influenza Avirus selected from the group consisting of influenza A virus H1, H2 andH5 subtypes, and thus it is useful for the prevention and treatment of adisease caused by the influenza A virus and is also useful for diagnosisof influenza A virus infection.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a set of graphs showing the binding affinities of CT109,CT111-1 and CT14-2 antibodies to monomeric Hemagglutinin (hereinafterreferred to as “HA”) and trimeric HA.

FIG. 2 is a set of graphs showing the binding affinities of CT104, CT120and CT123 antibodies to monomeric HA and trimeric HA.

FIG. 3 is a set of graphs showing the binding affinities of CT137, CT151and CT165 antibodies to monomeric HA and trimeric HA.

FIGS. 4A and 4B show vector maps of pCT145(A) and pCT147(B), in which Arepresents a pCT145 vector; B represents a pCT147 vector; pac: a genewhich encodes a Puromycin N-acetyl-tranferase (PAC); and DS representsdyad symmetry (EBNA1 binds to the dyad symmetry (DS) element in oriP ofEBV).

FIG. 5 is a map of an expression vector expressing the anti-influenza Avirus monoclonal antibody of the present invention.

FIGS. 6A to 6D show the results of animal (mouse) survival experimentsconducted using the anti-influenza A virus monoclonal antibody of thepresent invention, in which A represents a group injected with theantibodies 24 hours before challenging with H5N1 subtype virus(A/Vietnam/1203/04); B represents a group injected with the antibody 48hours after challenging with H5N1 subtype virus (A/Vietnam/1203/04); Crepresents a group injected with the antibody 24 hours before challeningwith pandemic H1N1 subtype virus (A/California/07/2009); and Drepresents a group injected with the antibody 24 hours beforechallenging with seasonal H1N1 subtype virus (A/puertoRico/8/1934).

FIG. 7 shows results of the virus titer-change in nasal wash of animal(ferret) experiments conducted using the CT120 of the present invention24 hours after challenging with H1N1 subtype (A/California/04/09).

FIG. 8 shows results of the virus titer-change in lung tissue of animal(ferret) experiments conducted using the CT120 of the present inventionafter challenging with H1N1 subtype (A/California/04/09).

DETAILED DESCRIPTION

Hereinafter, terms used herein will be defined as follows.

The term “influenza A viruses” refers to negative-strand and envelopedRNA(ribonucleic acid) virus belonging to the family Orthomyxoviridae.They have eight segments of single-stranded RNA and are classified asinfluenza types A, B and C. They are further divided into subtypes onthe basis of their major surface proteins HA (hemagglutinin) and NA(neuraminidase). Prior to the invention described herein, 16 Has and 9NAs were known.

The term “Hl subtype” used herein is intended to include H1N1, H1N2,H1N3, H1N4, H1N5, H1N6, H1N7, H1N8 and H1N9 of influenza A virus.

The term “H2 subtype” used herein is intended to include H2N1, H2N2,H2N3, H2N4, H2N5, H2N6, H2N7, H2N8 and H2N9 of influenza A virus.

The term “H5 subtype” used herein is intended to include H5N1, H5N2,H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9 of influenza A virus.

The term “hemagglutinin” (hereinafter referred to as “HA”) indicates theenvelope glycoprotein of influenza virus. HA mediates the adsorption andpenetration of influenza virus into a host cell. There are 16 known HAsubtypes.

The term “recovered or completely recovered patients” used herein refersto patients who were positive for influenza A virus due to influenza Avirus infection, but are negative for influenza A virus in blood after agiven period of time, indicating that the patients had recovered frominfection with influenza A virus.

Hereinafter, the present invention will be described in detail.

The present inventors isolated peripheral blood mononuclear cells(PBMCs) from blood collected from patients who had recovered frominfection with influenza A virus. Monoclonal antibody-producing B cellswere screened from the isolated PBMCs. The genetic information forproducing monoclonal antibodies in the screened B cells was obtained byan RT-PCR method and inserted into a pcDNA vector. The vector wastransfected into a CHO cell line to confirm preliminary antibodyproduction and it's HA-binding activity. A total of 82 antibodies werescreened. To more accurately measure binding affinity to HA, all theantibodies inserted into the pcDNA vector were transfected into humanF2N cells, and antibodies generated from the transfected cells werecomparatively analyzed by HA-ELISA using monomeric HA and trimeric HA asantigens, thereby 35 antibodies were selected, which were reacted withthe trimeric HA at higher degree than with the monomeric HA. The 35selected antibody genes in the pcDNA vectors were inserted into MarExexpression vectors, and then transfected into F2N cells to produce alarger amount of antibodies. These antibodies were used for amicroneutralization test (hereinafter referred to as an “MN test”) and ahemagglutination inhibition test (hereinafter referred to as an “HItest”) to determine the neutralizing activities against variousinfluenza viruses. A number of the antibodies exhibited high or lowneutralizing activities against various influenza viruses, but all theantibodies showed a negative reaction in the HI test. Through the MNtest, three monoclonal antibodies (CT104, CT120 and CT123 antibodies)showing neutralizing activity against various viruses were finallyselected. It was found that, among the three screened monoclonalantibodies, the CT104 had neutralizing activity against the H1 and H5subtypes, the CT120 had neutralizing activity against the H1, H2 and H5subtypes, and the CT123 had neutralizing activity against the H1 subtype(see Table 1). Also, in animal (mouse) survival experiments conductedusing the H1 and H5 subtype, the CT104 and the C120 exhibited excellentpreventive and therapeutic effects against H5N1 infection, and the threeantibodies all exhibited excellent preventive effects against pandemicand seasonal H1N1 infections (see FIG. 6). In another animal (ferret)experiments conducted using the H1 subtype, the CT120 exhibitedtherapeutic effects against H1N1 (A/California/04/09) infection (seeFIG. 7 and FIG. 8). Based on the above results, the present inventorshave completed an invention of neutralizing monoclonal antibodies whichprotect against influenza A virus infection.

Accordingly, the present invention provides an monoclonal antibodyhaving neutralizing activity against influenza A virus H1, H2 and H5subtypes.

In the present invention, the monoclonal antibody preferably binds to HAon the surface of influenza A virus. Also, the monoclonal antibody ispreferably derived from B cells present in the blood of patients who hadrecovered from infection with the influenza A virus H1N1 subtype.

In the present invention, the influenza A virus is preferably of theH1N1 subtype, and the influenza A virus HINI subtype is at least oneinfluenza virus selected from the group consisting ofA/Texas/05/2009-RG15, A/New York/18/2009-RG15, A/Solomon Islands/2006and A/Ohio/83. Also, the influenza A virus is preferably of the H2N2subtype, and the influenza A virus H2N2 subtype is A/Ann Arbor/6/60 ca.In addition, the influenza A virus is preferably of the H5N1 subtype,and the influenza A virus H5N1 subtype is one influenza virus selectedamong A/Vietnam/1203/04 and A/Anhui/1/05.

In the present invention, the monoclonal antibody has no neutralizingactivity against the influenza A virus H3N2 subtype.

The present invention also provides an anti-influenza A virus monoclonalantibody comprising the following light-chain and heavy-chainpolypeptide sequences, and a fragment and functional variant thereof:

a light chain comprising a CDR1 region comprising sequence(s) selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 7 and SEQ ID NO:12, a CDR2 region comprising a sequence of of SEQ ID NO: 2, and a CDR3region comprising sequence(s) selected from the group consisting of SEQID NO: 3, SEQ ID NO: 8 and SEQ ID NO: 13; and

a heavy chain comprising a CDR1 region comprising sequence(s) selectedfrom the group consisting of SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO:14, a CDR2 region comprising sequence(s) selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 10 and SEQ ID NO: 15, and a CDR3region comprising sequence(s) selected from the group consisting of SEQID NO: 6, SEQ ID NO: 11 and SEQ ID NO: 16.

The present invention also provides an anti-influenza A virus monoclonalantibody selected from the group consisting of the following monoclonalantibodies, and a fragment and functional variant thereof:

a monoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a sequence of SEQ ID NO: 1, a CDR2 region comprising asequence of SEQ ID NO: 2 and a CDR3 region comprising a sequence of SEQID NO: 3, and a heavy chain comprising a CDR1 region comprising asequence of SEQ ID NO: 4, a CDR2 region comprising a sequence of SEQ IDNO: 5, and a CDR3 region comprising a sequence of SEQ ID NO: 6;

a monoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a sequence of SEQ ID NO: 7, a CDR2 region comprising asequence of SEQ ID NO: 2 and a CDR3 region comprising a sequence of SETID NO: 8, and a heavy chain comprising a CDR1 region comprising asequence of SEQ ID NO: 9, a

CDR2 region comprising a sequence of SEQ ID NO: 10 and a CDR3 regioncomprising a sequence of SEQ ID NO: 11; and

a monoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a sequence of SEQ ID NO: 12, a CDR2 region comprising asequence of SEQ ID NO: 2 and a CDR3 region comprising a sequence of SEQID NO: 13, and a heavy chain comprising a CDR1 region comprising asequence of SEQ ID NO: 14, a CDR2 region comprising a sequence of SEQ IDNO: 15 and a CDR3 region comprising a sequence of SEQ ID NO: 16.

The monoclonal antibody preferably comprises a light chain comprising apolypeptide sequence of SEQ ID NO: 36, and a heavy chain comprising apolypeptide sequence of SEQ ID NO: 37. The monoclonal antibodypreferably has neutralizing activity against the influenza A virus H1and H5 subtypes and has no neutralizing activity against the influenza Avirus H3 subtype. The H1 subtype includes H1N1, H1N2, H1N3, H1N4, H1N5,H1N6, H1N7, H1N8 and H1N9, and the H5 subtype includes H5N1, H5N2, H5N3,H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9.

The monoclonal antibody preferably comprises a light chain comprising apolypeptide sequence of SEQ ID NO: 40, and a heavy chain comprising apolypeptide sequence of SEQ ID NO: 41. The monoclonal antibodypreferably has neutralizing activity against the influenza A virus H1,H2 and H5 subtypes and has no neutralizing activity against theinfluenza A virus H3 subtype. The H1 subtype includes H1N1, H1N2, H1N3,H1N4, H1N5, H1N6, H1N7, H1N8 and H1N9, and the H2 subtype includes H2N1,H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8 and H2N9. Also, the H5 subtypeincludes H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9.

The monoclonal antibody preferably comprises a light chain comprising apolypeptide sequence of SEQ ID NO: 44, and a heavy chain comprising apolypeptide sequence of SEQ ID NO: 45. The monoclonal antibodypreferably has neutralizing activity against the influenza A virus H1subtype and has no neutralizing activity against the influenza A virusH3 subtype. The H1 subtype includes H1N1, H1N2, H1N3, H1N4, H1N5, H1N6,H1N7, H1N8 and H1N9.

A fragment of the influenza A virus monoclonal antibody is not the wholeantibody, but is a portion of the antibody. It has the ability to bindto the influenza A virus HA, and is meant to include all the fragmentswhich bind to the HA competitively with the anti-influenza A virusmonoclonal antibody of the present invention.

In addition, also included are functional variants of the monoclonalantibody. If variants of the monoclonal antibody can complete with themonoclonal antibody of the present invention for binding specifically tothe influenza A virus HI, H2 and H5 subtypes, and fragments thereof,they are regarded as functional variants of the monoclonal antibody.Specifically, if functional variants can bind to the influenza A virusH1, H2 and H5 subtypes, or fragments thereof, and have neutralizingactivity against such subtypes or fragments, they are regarded as thefunctional variants. Functional variants include, but are not limitedto, derivatives that are substantially similar in primary structuralsequence, but which contain e.g. in vitro or in vivo modifications,chemical and/or biochemical, that are not found in the parent monoclonalantibody of the present invention. Such modifications include, forexample, acetylation, acylation, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, cross-linking, disulfide bond formation, glycosylation,hydroxylation, methylation, oxidation, pegylation, proteolyticprocessing, phosphorylation, and the like. Alternatively, functionalvariants can be monoclonal antibodies comprising an amino acid sequencecontaining substitutions, insertions, deletions or combinations thereofof one or more amino acids compared to the amino acid sequences of theparental monoclonal antibodies. Furthermore, functional variants cancomprise truncations of the amino acid sequence at either or both of theamino or carboxyl termini. Functional variants according to the presentinvention may have the same or different, either higher or lower,binding affinities compared to the parental monoclonal antibody but arestill capable of binding to the influenza A virus H1, H2 and H5subtypes, or fragments thereof. For example, functional variantsaccording to the invention may have increased or decreased bindingaffinities for the influenza A virus H1, H2 and H5 subtypes, orfragments thereof, compared to the parental binding molecules.Preferably, the amino acid sequences of the variable regions, including,but not limited to, framework regions, hypervariable regions, inparticular the CDR3 regions, are modified. Generally, the light-chain orheavy chain regions comprise three hypervariable regions, comprisingthree CDRs, and more conserved regions, the so-called framework regions(FRs). The hypervariable regions comprise amino acid residues from CDRsand amino acid residues from hypervariable loops. Functional variantsintended to fall within the scope of the present invention have at leastabout 50-99%, preferably at least about 60-99%, more preferably at leastabout 80-99%, even more preferably at least about 90-99%, in particularat least about 95-99%, and in particular at least about 97-99% aminoacid sequence homology with the parental monoclonal antibody as definedherein. Computer algorithms such as Gap or Bestfit known to a personskilled in the art can be used to optimally align amino acid sequencesto be compared and to define similar or identical amino acid residues.Functional variants can be obtained either by altering the parentalmonoclonal antibodies or parts thereof by general molecular biologymethods known in the art including PCR, oligonucleotide-directedmutagenesis and site-directed mutagenesis, or by organic syntheticmethods.

The present invention also provides an anti-influenza A virus monoclonalantibody comprising the following light-chain and heavy-chainpolynucleotide sequences, and a fragment and functional variant thereof:

a light chain comprising a CDR1 region comprising sequence(s) selectedfrom the group consisting of SEQ ID NO: 17, SEQ ID NO: 23 and SEQ ID NO:28, a CDR2 region comprising a sequence of SEQ ID NO: 18 or SEQ ID NO:29, and a CDR3 region comprising sequence(s) selected from the groupconsisting of SEQ ID NO: 19, SEQ ID NO: 24 and SEQ ID NO: 30; and

a heavy chain comprising a CDR1 region comprising sequence(s) selectedfrom the group consisting of SEQ ID NO: 20, SEQ ID NO: 25 and SEQ ID NO:31, a CDR2 region comprising sequence(s) selected from the groupconsisting of SEQ ID NO: 21, SEQ ID NO: 26 and SEQ ID NO: 32, and a CDR3region comprising sequence(s) selected from the group consisting of SEQID NO: 22, SEQ ID NO: 27 and SEQ ID NO: 33.

The present invention also provides an anti-influenza A virus monoclonalantibody selected from the group consisting of the following monoclonalantibodies, and a fragment and functional variant thereof:

a monoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a sequence of SEQ ID NO: 17, a CDR2 region comprising asequence of SEQ ID NO: 18 and a CDR3 region comprising a sequence of SEQID NO: 19, and a heavy chain comprising a CDR1 region comprising asequence of SEQ ID NO: 20, a CDR2 region comprising a sequence of SEQ IDNO: 21 and a CDR3 region comprising a sequence of SEQ ID NO: 22;

a monoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a sequence of SEQ ID NO: 23, a CDR2 region comprising asequence of SEQ ID NO: 18 and a CDR3 region comprising a sequence of SEQID NO: 24, and a heavy chain comprising a CDR1 region comprising asequence of SEQ ID NO: 25, a CDR2 region comprising a sequence of SEQ IDNO: 26 and a CDR3 region comprising a sequence of SEQ ID NO: 27; and

a monoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a sequence of SEQ ID NO: 28, a CDR2 region comprising asequence of SEQ ID NO: 29 and a CDR3 region comprising a sequence of SEQID NO: 30, and a heavy chain comprising a CDR1 region comprising asequence of SEQ ID NO: 31, a CDR2 region comprising a sequence of SEQ IDNO: 32 and a CDR3 region comprising a sequence of SEQ ID NO: 33.

In the present invention, the monoclonal antibody preferably comprises alight chain comprising a polynucleotide sequence of SEQ ID NO: 34, and aheavy chain comprising a polynucleotide sequence of SEQ ID NO: 35. Themonoclonal antibody preferably has neutralizing activity against theinfluenza A virus H1 and H5 subtypes and has no neutralizing activityagainst the influenza A virus H3N2 subtype. The H1 subtype includesH1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8 and H1N9, and the H5subtype includes H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 andH5N9.

In the present invention, the monoclonal antibody preferably comprises alight chain comprising a polynucleotide sequence of SEQ ID NO: 38, and aheavy chain comprising a polynucleotide sequence of SEQ ID NO: 39. Themonoclonal antibody preferably has neutralizing activity against theinfluenza A virus H1, H2 and H5 subtypes and has no neutralizingactivity against the influenza A virus H3N2 subtype. The H1 subtypeincludes H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8 and H1N9, andthe H2 subtype includes H2N1, H2N2, H2N3, H2N4, H2N5, H2N6, H2N7, H2N8and H2N9. Also, the H5 subtype includes H5N1, H5N2, H5N3, H5N4, H5N5,H5N6, H5N7, H5N8 and H5N9.

In the present invention, the monoclonal antibody preferably comprises alight chain comprising a polynucleotide sequence of SEQ ID NO: 42, and aheavy chain comprising a polynucleotide sequence of SEQ ID NO: 43. Themonoclonal antibody preferably has neutralizing activity against theinfluenza A virus H1 subtype and has no neutralizing activity againstthe influenza A virus H3 subtype. The H1 subtype includes H1N1, H1N2,H1N3, H1N4, H1N5, H1N6, H1N7, H1N8 and H1N9.

The present invention also provides an isolated nucleic acid moleculeencoding said anti-influenza A virus monoclonal antibody.

The nucleic acid molecule of the present invention includes all nucleicacid molecules obtained by “translating” the amino acid sequences of theantibodies of the present invention to polynucleotide sequencesaccording to methods known to a person skilled in the art. Accordingly,various polynucleotide sequences with open reading frames (ORFs) can beprepared and are also included in the scope of the nucleic acidmolecules of the present invention.

The present invention also provides an expression vector containing saidnucleic acid molecule inserted therein. The expression vector canpreferably be derived from one selected from the group consisting of,but not limited to, an MarEx expression vector produced by CelltrionInc. (Korea), a commercially widely available pCDNA vector, F, R1, RP1,Col, pBR322, ToL, Ti vector; cosmids; phages such as lambda, lambdoid,M13, Mu, Pl, P22, Q[i, T-even, T2, T4, T7, etc; plant viruses. Any of avariety of expression vectors known to those skilled in the art can beused in the present invention, and the choice of the expression vectoris dependent on the nature of the host cell of choice. Introduction ofthe vector in host cells can be effected by, but not limited to, calciumphosphate transfection, virus infection, DEAE-dextran mediatedtransfection, lipofectamin transfection or electroporation, and anyperson skilled in the art can select and use an introduction methodsuitable for the expression vector and host cell used. Preferably, thevector contains one or more selection markers, but is not limitedthereto, and a vector containing no selection marker may also be used.The choice of the selection markers may depend on the host cells ofchoice, although this is not critical to the present invention as iswell known to persons skilled in the art.

To facilitate the purification of the nucleic acid molecule of thepresent invention, a tag sequence may be inserted into the expressionvector. Examples of the tag include, but are not limited to, ahexa-histidine tag, a hemagglutinin tag, a myc tag or a FLAG tag. Any oftags facilitating purification, known to those skilled in the art, maybe used in the present invention.

The present invention also provides an anti-influenza A virus monoclonalantibody-producing cell line transformed with said expression vector.

In the present invention, the cells include, but are not limited to, themammalian cell, the plant cell, the insect cell, the fungal cell or thebacterial origin cell. As for the mammalian cell, one selected from thegroup consisting of, but not limited to, CHO cell, F2N cell, CSO cell,BHK cell, Bowes melanoma cell, HeLa cell, 911 cell, AT1080 cell, A549cell, HEK 293 cell and HEK293T cell, may preferably be used as a hostcell. Any cell usable as mammalian host cell known to those skilled inthe art may be used in the present invention.

The present invention also provides a method of screening an antibodyhaving a neutralizing activity against influenza A virus in patientsrecovered from infection with influenza A virus, the method comprisingthe steps of: 1) examining whether patients infected with influenza Avirus is completely recovered, and screening patients, who are negativefor influenza A virus in blood, from the examined patients; 2)collecting blood from the completely recovered patients screened in step1); 3) isolating B cells from the patient's blood collected in step 2);4) screening B cells, which produce an HA-binding antibody, from the Bcells isolated in step 3); 5) extracting RNAs from the B cells screenedin step 4); 6) amplifying antibody genes from the RNAs extracted in step5); 7) cloning the genes amplified in step 6) into expression vectors;8) transfecting the expression vectors of step 7) into host cells; 9)examining whether the transfected host cells of step 8) produce theHA-binding antibody; 10) culturing the screened transfected cell of step9); 11) purifying antibodies binding to the HA of influenza A virus fromthe transfected cell cultures of step 10); 12) re-confirming whether theantibodies purified in step 11) have neutralizing activity againstinfluenza A virus; and 13) re-screening an antibody confirmed to haveneutralizing activity against influenza A virus in step 12).

The present invention also provides a composition comprising saidanti-influenza A virus monoclonal antibody.

The composition of the present invention may contain, in addition to theanti-influenza A virus monoclonal antibody, a pharmaceuticallyacceptable excipient. Pharmaceutically acceptable excipients are wellknown to those skilled in the art.

The present invention also provides a composition for preventing andtreating a disease caused by influenza A virus, comprising saidanti-influenza A virus monoclonal antibody.

The composition of the present invention may contain, in addition to theanti-influenza A virus monoclonal antibody, a pharmaceuticallyacceptable excipient. Pharmaceutically acceptable excipients are wellknown to those skilled in the art.

Also, the preventive and therapeutic composition of the presentinvention may comprise at least five other therapeutic agents forinfluenza A. The preventive and therapeutic composition of the presentinvention may comprise various monoclonal antibodies binding to theinfluenza A virus H1, H2 and H5 subtypes or fragments thereof, whereinthe monoclonal antibodies can exhibit a synergistic effect onneutralizing activity. Also, the preventive and therapeutic compositionof the present invention may additionally comprise one or more othertherapeutic agents or diagnostic agents. The therapeutic agents include,but are not limited to, anti-viral drugs. Such drugs may includeantibodies, small molecules, organic or inorganic compounds, enzymes,polynucleotide sequences, anti-viral peptides, etc.

The preventive and therapeutic composition of the present invention mustbe sterile and stable under the conditions of manufacture and storage.Also, it can be in powder form for reconstitution in the appropriatepharmaceutically acceptable excipient before or at the time of delivery.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying that yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. Alternatively, the composition of the presentinvention can be in solution and the appropriate pharmaceuticallyacceptable excipient can be added and/or mixed before or at the time ofdelivery to provide a unit dosage injectable form. Preferably, thepharmaceutically acceptable excipient used in the present invention issuitable to high drug concentration, can maintain proper fluidity and,if necessary, can delay absorption.

The choice of the optimal route of administration of the preventive andtherapeutic composition will be influenced by several factors includingthe physico-chemical properties of the active molecules within thecomposition, the urgency of the clinical situation and the relationshipof the plasma concentrations of the active molecules to the desiredtherapeutic effect. For example, the monoclonal antibodies of thepresent invention can be prepared with carriers that will protect themagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers, such as ethylene vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid, may be used in the present invention. Furthermore,the monoclonal antibody may be coated or co-administered with a materialor compound that prevents the inactivation of the antibody. For example,the monoclonal antibody may be administered together with an appropriatecarrier, for example, liposome or a diluent.

The routes of administration of the preventive and therapeuticcomposition of the present invention can be divided into oral andparenteral administration. The preferred administration route isintravenous, but is not limited thereto.

Oral dosage forms can be formulated as tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsions,hard capsules, soft gelatin capsules, syrups or elixirs, pills, dragees,liquids, gels, or slurries. These formulations can containpharmaceutical excipients including, but not limited to, granulating anddisintegrating agents, binding agents, lubricating agents,preservatives, coloring, flavoring or sweetening agents, vegetable ormineral oils, wetting agents, and thickening agents.

Formulations for parenteral administration can be in the form of aqueousor non-aqueous isotonic sterile non-toxic injection or infusionsolutions or suspensions. The solutions or suspensions may compriseagents that are non-toxic to recipients at the dosages andconcentrations employed such as 1,3-butanediol, Ringer's solution,Hank's solution, isotonic sodium chloride solution, oils, fatty acids,local anaesthetic agents, preservatives, buffers, viscosity orsolubility increasing agents, water-soluble antioxidants, oil-solubleantioxidants and metal chelating agents.

The present invention provides a composition for diagnosis of influenzaA virus, which comprises a conjugate comprising a tag conjugated to saidanti-influenza A virus monoclonal antibody.

The diagnostic composition of the present invention comprises at leastone detectable tag, such as a detectable moiety/agent. The tag can beconjugated non-covalently to the monoclonal antibody of the presentinvention. The tag can also be conjugated directly to the monoclonalantibody through covalent bonding. Alternatively, the tag can beconjugated to the monoclonal antibody by means of one or more linkingcompounds. Techniques for conjugating the tag to the monoclonal antibodyare well known to those skilled in the art. The detectable moiety/agentas the tag is preferably one selected from the group consisting of, butnot limited to, enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,positron emitting metals, and non-radioactive paramagnetic metal ions.

The present invention also provides a method of treating a diseasecaused by influenza A virus, the method comprising a step ofadministering an influenza A virus monoclonal antibody of the presentinvention to a subject having a disease caused by influenza A virus.

In the therapeutic method of the present invention, the influenza Avirus is preferably at least one selected from the group consisting ofH1, H2 and H5 subtypes. The H1 subtype includes H1N1, H1N2, H1N3, H1N4,H1N5, H1N6, H1N7, H1N8 and H1N9, and the H2 subtype includes H2N1, H2N2,H2N3, H2N4, H2N5, H2N6, H2N7, H2N8 and H2N9. Also, the H5 subtypeincludes H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9.

In the therapeutic method of the present invention, any therapeuticagent for disease caused by influenza A virus known to those skilled inthe art may be administered together with the monoclonal antibody of thepresent invention.

In the therapeutic method of the present invention, the disease causedby influenza A virus may be one selected from the group consisting of,but not limited to, a new strain offlu, pandemic flu and seasonal flu.

In the therapeutic method of the present invention, the dose of theinfluenza A virus monoclonal antibody may be adjusted to the optimumresponse. The dose is, for example, 0.01-200 mg/kg, preferably 0.1-150mg/kg, and more preferably 1-100 mg/kg, but is not limited thereto.Several divided doses may be administered daily, or the dose may beproportionally reduced as indicated by the exigencies of an individual'ssituation. The composition of the present invention may be administeredin a single serving or in multiple servings spaced throughout the day.The mode of administration is not limited in the present invention andcan be decided by the attending physician.

In the therapeutic method of the present invention, the routes ofadministration of the influenza A virus monoclonal antibody can bedivided into oral administration and parenteral administration. Thepreferred administration route is intravenous, but is not limitedthereto.

The present invention also provides a method for preventing a diseasecaused by influenza A virus, the method comprising a step ofadministering an influenza A virus monoclonal antibody of the presentinvention to a subject.

In the preventive method of the present invention, the influenza A virusmonoclonal antibody may be administered together with any preventiveagent for disease caused by influenza A virus known to those skilled inthe art.

In the preventive method of the present invention, the dose of theinfluenza A virus monoclonal antibody may be adjusted to the optimumresponse. The dose is, for example, 0.01-200 mg/kg, preferably 0.1-150mg/kg, and more preferably 1-100 mg/kg, but is not limited thereto.Several divided doses may be administered daily, or the dose may beproportionally reduced as indicated by the exigencies of an individual'ssituation. The composition of the present invention may be administeredin a single serving or in multiple servings spaced throughout the day.The mode of administration is not limited, and can be decided by theattending physician.

The present invention also provides a method for diagnosis of influenzaA virus infection in a patient, the method comprising the steps of: 1)contacting a sample with the anti-influenza A virus monoclonal antibodyof the present invention; and 2) detecting a reaction between themonoclonal antibody and the sample. Alternatively, the diagnostic methodmay comprise the steps of: 1) contacting a sample with a diagnosticcomposition of the present invention; and 2) detecting a reactionbetween the diagnostic composition and the sample.

In the diagnostic method of the present invention, the influenza A virushas one or more subtype(s) selected from the group consisting of H1, H2and H5. The H1 subtype includes H1N1, H1N2, H1N3, H1N4, H1N5, H1N6,H1N7, H1N8 and H1N9, and the H2 subtype includes H2N1, H2N2, H2N3, H2N4,H2N5, H2N6, H2N7, H2N8 and H2N9. Also, the H5 subtype includes H5N1,H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9.

In the diagnostic method of the present invention, the monoclonalantibody of the present invention may, if necessary, be conjugated witha tag for diagnosis and detection according to any method known to aperson skilled in the art.

In the diagnostic method of the present invention, the sample ispreferably one selected from the group consisting of, but not limitedto, phlegm, spittle, blood, lung cell, lung tissue mucus, respiratorytissue and salvia. The sample can be prepared according to anyconventional method known to a person skilled in the art.

In the diagnostic method of the present invention, the method fordetecting the reaction may be one selected from the group consisting of,but not limited to, homogeneous and heterogeneous binding immunoassays,such as radio-immunoassays (RIA), ELISA, immunofluorescence,immunohistochemistry, FACS, BIACORE and Western blot analyses. Anydetection method known to a person skilled in the art may be used in thepresent invention.

The present invention also provides a kit for diagnosis of influenza Avirus, which comprises: 1) an anti-influenza A virus monoclonal antibodyof the present invention; and 2) a container.

In addition, the present invention provides a kit for diagnosis ofinfluenza A virus infection, which comprises: 1) a composition fordiagnosis of influenza A virus infection according to the presentinvention; and 2) a container.

In the diagnostic kit of the present invention, the influenza A virushas preferably one or more subtype(s) selected from the group consistingof H1, H2 and H5. The H1 subtype includes HIN1, H1N2, H1N3, H1N4, H1N5,H1N6, H1N7, H1N8 and H1N9, and the H2 subtype includes H2N1, H2N2, H2N3,H2N4, H2N5, H2N6, H2N7, H2N8 and H2N9. Also, the H5 subtype includesH5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9.

In the diagnostic kit of the present invention, a solid support isincluded in the container 2). The monoclonal antibody of the presentinvention can be attached to a solid support, and this solid support maybe porous or nonporous, planar or non-planar.

EXAMPLES Example 1: Isolation of PBMC from Blood from Patients WhoRecovered from Flu

A recovered patient group consisted of patient volunteers who were 2-4weeks after confirmation of new flu infections. The volunteers wereconfirmed to have no influenza virus (H1N1) in their blood and had anantibody against the new influenza virus. This study was performed underthe approval of the Institutional Review Board (IRB). This patientsgroup had the following characteristics: (1) the patients were notvaccinated against seasonal flu; (2) the patients were negative forother infectious viruses, that is, HBsAg, and were negative for anti-HCVantibody and anti-HIV antibody; (3) the patients were negative(identified by RT-PCR) for the influenza virus H1N1 subtype in plasma;(4) the patients showed a titer of 1:160 or higher in serum in ELISAassays for the (monomeric) HA(H1N1) of the influenza A virus H1N1subtype. About 100 ml of whole blood was collected from the volunteers,and peripheral blood mononuclear cells (PBMCs) were isolated from thecollected blood using Lymphoprep™ (Axis-Shield, Norway, 1114545). Theisolated PBMCs were washed three times with phosphate-buffered saline,suspended at 2×10⁷ cells/me in KM banker II freezing medium (Cosmobio,Japan, KOJ-16092010), and stored in a liquid nitrogen tank.

Example 2: Primary Screening of Monoclonal Antibodies

B cells secreting antigen-specific antibodies were screened using themethod described by Jin et al. (Tin A. et al., 2009. Nat Med. 15,1088-1092). Briefly, the PBMCs were added to each well of the preparedmicroarray chip at a density of one cell/well. Antibodies secreted fromthe single cells were confirmed by the precoated anti-human IgGantibody. Whether the screened antibody-secreting cells secretedHA-binding antibodies was examined using the labeled HA antigen by anenzyme-linked immunospot assay (ELISPOT; Sedgwick J. D., 2005, MethodsMol Bio. Vol. 302, pp. 314). The complete sequences of the heavy-chainand light-chain genes of the antibodies from the individualantibody-secreting cells were obtained by a reversetranscription-polymerase chain reaction (RT-PCR). The obtainedheavy-chain and light-chain DNAs were inserted into pcDNA 3.1(+)expression vectors (Invitrogen, USA, V790-20) to prepare expressionvectors producing each of the heavy chain and light chain of theantibodies. The prepared expression vectors were co-transfected into CHOcells. After that, using the antibodies derived from the transfected CHOcells, 82 antibodies binding to HA were primarily selected through theHA-ELISA described in Example 3 below. Herein, all the antibodiesshowing a reaction with HA were primarily screened without seriallydiluting the antibody samples.

Example 3: Second Step Screening of Monoclonal Antibodies and theirProduction

In order to secondarily screen monoclonal antibodies having high bindingaffinity for recombinant HA from the 82 primarily screened antibodies,HA-ELISA was performed using monomeric HA and trimeric HA. Recombinantmonomeric HA (11055-VO8H) from influenza A virus (A/California/04/2009)was purchased from Sino Biological Inc. (China). The purchased monomericHA consisted of an extra-cellular domain (met 1-gln529) of HA comprising10 polyhistidine residues at the C-terminus and was derived fromtransfected human cells. Recombinant trimeric HA (FR-180) was providedby IRR (Influenza Reagent Resource, USA). The trimeric HA from H1N1(A/California/04/2009) included a thrombin cleavage site at theC-terminus, a trimerizing domain (foldon) and six histidine residues andwas produced using a baculovirus system.

The reactivity of the antibody with the HA antigen was measured by ELISAusing the HA and the antibody. Specifically, first, 50 μl of each ofmonomeric HA or trimeric HA (250 ng/rne) was coated onto each well of a96-well microtiter plate (Nunc, Denmark, 449824). The plate was blockedwith phosphate-buffered saline (Teknova, USA, D5120) containing 1%bovine serum albumin (BSA), and then a 3-fold serially diluted antibodysample (starting concentration: 1 μg/ml) was added to each well of theplate. Next, the plate was incubated at room temperature for 1 hour andthen treated with peroxidase-labeled goat anti-human gamma antibody(Zymed, USA, 62.8420). After incubation for 1 hour at room temperature,the plate was incubated with tetramethylbenzydine (TMB; Sigma-Aldrich,USA, T0440), and the incubation was stopped by adding 1 N HCl. Theabsorbance at 450/570 nm was measured using a plate reader (Spectramaxplus 384, Molecular Device), and the antigen-antibody reactivity wasgraphically expressed using Graphpad prism program (GraphPad SoftwareInc. USA).

As shown in FIG. 1, the CT109, CT111-1 and CT154-2 antibodies showedvery high binding activities against the trimeric HA and also showedhigh binding activities against the monomeric HA, but lower than thebinding activities against the trimeric HA. Also, the CT104, CT120 andCT123 antibodies showed high binding activities against the trimeric HA,but showed little or no binding activities against the monomeric HA(FIG. 2). Other antibodies (CT137, CT151 and CT165 antibodies) showedlittle or no binding activities against the two antigens (FIG. 3).

On the basis of the results shown in FIGS. 1 to 3, from the 82 primarilyscreened antibodies, 35 antibodies showing high binding activitiesagainst the trimeric HA were secondarily selected. To quantitate thebinding activities of the monoclonal antibodies and thus narrow down thenumbers of monoclonal antibodies in MN test, it was necessary toincrease the expression levels of the secondarily selected antibodies.Therefore, these antibody genes were recloned from the cDNA vectors intoMarEx expression vectors constructed and patented by Celltrion, Inc., inthe following manner. After recloning, the MarEx expression vectorscontaining the antibody genes were used to produce antibodies requiredfor a MN test and a HI test.

The original pcDNA vectors containing each of the heavy-chain genes andlight-chain genes of the 35 secondarily selected antibodies were treatedwith the restriction enzymes Nhe I and Pme I to separate heavy-chaingenes and light-chain genes. The obtained heavy-chain genes andlight-chain genes were respectively inserted into pCT145 vectors andpCT147 vectors, which had been treated with the same restrictionenzymes. The pCT145 and pCT147 vectors were constructed by Celltrion,Inc., in order to construct the heavy chain and the light chainexpressing vectors, respectively (FIG. 4). Next, in order to constructexpression vectors containing a heavy-chain transcription unit(promoter-heavy chain gene-poly A) together with a light-chaintranscription unit (promoter-light chain gene-poly A), the pCT145vectors containing the heavy-chain genes were treated with therestriction enzymes Pac I and Asc I to separate heavy-chaintranscription units, and then the pCT147 vectors containing thelight-chain genes were treated with the same restriction enzymes andinserted with the separated heavy-chain transcription units. Then,vectors containing the heavy-chain transcription unit together with thelight-chain transcription unit were screened using restriction enzymes(FIG. 5). The screened vectors were extracted using an Endofree plasmidmaxi kit (QIAGEN, Germany, 12362), and the nucleotide sequences wereanalyzed using the part of the extracted DNA samples, therebydetermining the nucleotide sequences of the antibodies.

Next, the DNA of the extracted antibodies was transfected intosuspension cell of F2N cell line (constructed by Celltrion, Inc.,Korea), to produce monoclonal antibodies in transient production manner.Herein, the transfection was performed in the following manner.Transfection of the cells with plasmid DNA was carried out using thecationic polymer FreeStyle™ Max (Invitrogen, USA, 16447-100) accordingto the manufacturer's instruction. On the day before transfection, theF2N cells cultured in EX-CELL 293 serum-free media (SAFC, LIK, 14571C;hereinafter referred to as “EX-CELL 293 media”) were centrifuged andsuspended at a cell concentration of 1×10⁶ cells/ml in modified EX-CELL293 medium (SAFC, LIK, 65237; made to order), and 80 ml of the cellsuspension was seeded into a 250 ml Erlenmeyer flask, or 200 ml of thecell suspension was seeded into a 1 1 Erlenmeyer flask in an amount of200 ml. On the day of transfection, in the case in which 80 ml of thecell suspension was seeded, each of DNA encoding a monoclonal antibodyand 100 μl of FreeStyle™ Max reagent was diluted to a volume of 1.6 mlusing OptiPRO SFM II medium (Invitrogen, USA, 12309) and stirred gently.In the case in which 200 ml of the cell suspension was seeded, each of250 μg of DNA and 250 μg of FreeStyle™ Max reagent was diluted to avolume of 4 ml using OptiPRO SFM II medium and stirred gently.Immediately after the stirring process, the solution containingFreeStyle™ Max reagent diluted therein was mixed with the solutioncontaining DNA diluted therein, and the mixed solution was incubated atambient temperature for 19 minutes. During the incubation process atambient temperature for 19 minutes, the seeded F2N cells were diluted toa cell concentration of 0.8×10⁶ cells using fresh modified EX-CELL 293medium. After incubation for 19 minutes, the mixed solution of DNA andFreeStyle™Max reagent was added to the F2N cell culture prepared fortransfection. On the day after transfection, the same amount of EX-CELL293 medium was added to the transfected cells, which were then culturedfor 7-8 days, thereby producing monoclonal antibodies.

Example 4: Examination of In Vitro Neutralizing Activity Against Viruses

From the screening of 35 monoclonal antibodies, 11 antibodies whichshowed high binding affinities to the trimeric HA in HA-ELISA wereselected and subjected to a MN test in order to examine theirneutralizing activity against various influenza viruses.

Example 4-1 Culture of MDCK Cell Line and Determination of VirusConcentration

As Madin-Darby canine kidney (MDCK) cell line, the London line (MDCK-L)was used. The MDCK cell line was cultured in a 5% CO₂ humidifiedincubator at 37° Cusing a DMEM medium (Gibco, USA, 11965) containing 10%FBS (Atlas Biologicals, USA, F0500A), 1× penicillin/streptomycin (Gibco,USA, 15140), 25 mM HEPES (Gibco, USA, 15630) and 2 mM L-glutamine(Gibco, USA, 25030).

Virus concentration was quantified by ELISA to determine the mediantissue culture infective dose (TCID₅₀). The determination of virusconcentration was performed in the following manner. First, a virusstock was serially diluted 10-fold with a virus diluent [DMEM (Gibco,USA), 3% BSA (Gibco, USA, 15260), 1× pecinillin/streptomycin (Gibco,USA), and 25 mM HEPES (Gibco, USA)], and 100 μl of the diluted virus wasadded to each well of a 96-well plate. As a negative control, a virusdiluent containing no virus was used. Then, the MDCK cell line which wasbeing cultured was treated with trypsin, separated from the cultureincubator, and then treated with MDCK culture medium to neutralizetrypsin. Next, the cell pellets were washed twice withphosphate-buffered saline, and then diluted to a cell concentration of5×10⁵ cells/ml with a virus diluent. 3-4 μg/ml of TPCK-trypsin (Sigma,USA) was added to the 96-well plate containing the virus, and thenimmediately, 100 μl of the MDCK cell line was added to each well of theplate and incubated in a 5% CO₂ humidified incubator at 37° C. for 20hours. The incubated plate was washed once with phosphate bufferedsaline, and then 200 μl of a mixed solution of cold acetone: phosphatebuffered saline (PBS) (80:20) was added to each well of the plate. Next,the cells were fixed for 8 minutes, and then the plate was dried atambient temperature for 20 minutes. 200 μl of phosphate buffered salinewas added to each well of the plate to wash each well twice. 100 μl ofbiotinylated anti-nuclear protein (NP) monoclonal antibody (Milipore,USA, MAB8257B), which was diluted 2,000-fold with 1% BSA-containingphosphate buffered saline, was added to each well of the plate andincubated at ambient temperature for 1 hour. The plate was washed threetimes with 200 μl/well of phosphate buffered saline, and astreptavidin-HRP-conjugated antibody was diluted 20,000-fold with 1%BSA-containing phosphate buffered saline. Then, 100 μl of the antibodydilution was added to each well of the plate and incubated at roompressure for 1 hour. After washing the plate four times with phosphatebuffered saline, 100 μl of OPD solution (Sigma, USA, P8287) was added toeach well, and the plate was developed at room temperature for 10minutes. The plate was treated with 50 μl/well of 3 M HCl to stop thecolor development, and then the OD₄₉₀ of each well was measured. Basedon the measured OD₄₉₀, TCID_(so) was calculated using the method of Reed& Muench (The American 1938).

Example 4-2: MN Assay

Each antibody was diluted to a concentration of 10 μg/ml with a virusdiluent. From this initial concentration, the antibody dilution wasserially diluted 2-fold with a virus diluent, and 50 μl of the dilutionwas added to each well of a 96-well plate. Also, 50 j of viruses wereadded to each well of the plate at a concentration corresponding to 100TCID₅₀ and were incubated in a 5% CO₂ humidified incubator at 37° C. for1 hour. Next, 3-4 μg/ml of TPCK-trypsin (Sigma, USA, T1426) was added toeach well, and 100 of the treated MDCK cells was added to each well, andthen incubated in a 5% CO₂ humidified incubator at 37° C. for 20 hours.Then, an MN assay was carried out according to the same method as thevirus quantification method described in Example 4-1, thus determiningthe OD₄₉₀ value of each well. The wells showing OD₄₉₀ values higher thanthat of the well introduced only with the cells was determined to beinfected with viruses. Among OD₄₉₀ values for each antibody at which novirus antigen was detected, the lowest concentration (μg/ml) of theantibody is shown in Table 1, and the lower concentration of theantibody means the higher neutralizing activity against virus.

TABLE 1 Results of Micromeutralization assay (MN assay) carried outusing screened antibodies and viruses of various types H1 Pandemic(A/New H1 Seasonal H2 H5 H3 (A/Texas/05/2009- York/18/2009- (A/Solomon(A/Ann (A/Viet- (A/Wiscon- mab ID RG15) RG18) Islands/2006) (A/Ohio/83)Arbor/ 6/60 ca) nam/1203/04) (A/Anhui/1/05) sin/67/2005) CT104 0.3130.625 0.625 0.313 >10 1.25 0.625 >10 CT105 1.25 5.0 >10 2.5 >10 >1010 >10 CT109 >10 >10 >10 >10 >10 >10 >10 >10CT111-1 >10 >10 >10 >10 >10 >10 >10 >10 CT112-1 0.625 1.25 5.0 0.625 >105 2.5 >10 CT113 1.25 1.25 1.25 0.625 >10 5 2.5 >10 CT117 2.5 2.5 5.02.5 >10 10 10 >10 CT119 1.25 2.5 5.0 1.25 >10 10 2.5 >10 CT120 0.3130.313 0.625 0.156 2.5 1.25 0.625 >10 CT122-1 2.510 >10 >10 >10 >10 >10 >10 CT123 0.313 0.625 1.250.313 >10 >10 >10 >10 * unit: μg/ml

As can be seen from the results of MN assays of 11 candidate antibodiesagainst H1, H2, H3 and H5 subtype influenza viruses, the CT104 showedneutralizing activities against two pandemic H1N1 subtype viruses(A/Texas/05/2009 and A/New York/18/2009) and two seasonal H1N1 subtypeviruses (A/Solomon Islands/3/2006 and A/Ohio/83) at low concentrations(0.313-0.625 μg/ml) and also neutralized two H5N1 subtype viruses(A/Vietnam/1203/04 and A/Anhui/1/05) at concentrations of 1.25 μg/ml and0.625 μg/ml, respectively. However, the CT104 antibody did not showneutralizing activity against the H2N2 subtype virus (A/AnnArbor/6/60ca) and the H3N2 subtype virus (A/Wisconsin/67/2005). TheCT123 showed neutralizing activity only against four H1N1 subtypeviruses tested. Particularly, the CT120 antibody showed highneutralizing activity against the four H1N1 subtype influenza viruses,one H2N2 influenza subtype (A/Ann Arbor/6/60 ca) and two H5N1 subtypeinfluenza viruses. However, the above-described antibodies did not showneutralizing activity against the H3N2 subtype belonging to the H3Glade.

The IC₅₀ values of the three screened antibodies having neutralizingactivity against viruses were measured for comparison, and themeasurement results are shown in Table 2 below. Herein, the IC₅₀ valueis the antibody concentration at which the antibody shows 50% of thehighest neutralizing activity against viruses, and the lower value ofIC₅₀ means the higher neutralizing activity of the antibody.

TABLE 2 IC50 values of neutralizing activities of CT104, CT120 and CT123against two types of pandemic H1N1 viruses A/Texas/05/2009-RG15 A/NewYock/18/2009-RG18 Antibody con- Antibody con- Antibody centration* IC₅₀centration* IC₅₀ CT104 0.313 μg/ml 0.29 μg/ml 1.25 μg/ml 0.56 μg/mlCT120 0.156 μg/ml 0.15 μg/ml 0.313 μg/ml  0.31 μg/ml CT123 0.625 μg/ml0.068 μg/ml  1.25 μg/ml 0.29 μg/ml Note. Antibody concentration* is aneutralizing antibody concentrations shown in Table 1.

As can be seen in Table 2 above, the three antibodies had very low IC₅₀values, and thus had high neutralizing activity against the two virusesshown in Table 2.

Example 5: Examination of the Ability of Antibody to Inhibit aHemagglutination Reaction Caused by Viruses

An antibody was serially diluted 2-fold on a V-bottom 96-well plate, andviruses of 4-fold HA unit were added to and mixed with the antibody.Next, the plate was incubated at room temperature for 30 minutes, andthen 1% avian red blood cells were added to each well of the plate. Thehemagglutination inhibition end point was determined as the lowestantibody concentration in which no hemagglutination-reaction wasobserved.

As a result, all the antibodies tested did not inhibit hemagglutinationfor two types of pandemic H1N1 subtype viruses (A/Texas/05/2009-RG15 andA/New York/18/2009-RG18) even at high concentrations (>20 μg/ml) (Table3).

TABLE 3 Results of Hemagglutination-inhibition test for screenedantibodies against two types of pandemic H1N1 viruses AntibodyA/Texas/05/2009-RG15 A/New Yock/18/2009-RG18 CT104 >20 μg/ml >20 μg/mlCT105 >20 μg/ml >20 μg/ml CT109 >20 μg/ml >20 μg/ml CT111-1 >20μg/ml >20 μg/ml CT112-1 >20 μg/ml >20 μg/ml CT113 >20 μg/ml >20 μg/mlCT119 >20 μg/ml >20 μg/ml CT120 >20 μg/ml >20 μg/ml CT122-1 >20μg/ml >20 μg/ml CT123 >20 μg/ml >20 μg/ml

Example 6: Examination of Preventive and Therapeutic Effects ofAntibodies on Influenza Viruses Infection by Animal Experiment Example6-1: Mouse Survival Experiment

In order to examine the CT104, CT120 and CT123 antibodies screened inthe above Examples have preventive and therapeutic effects against H1N1and H5N1 subtype viruses in mice, the following experiment was carriedout.

Each group consisting of five mice that were nasally infected with10×LD₅₀ of viruses. Each of the three screened antibodies (CT-104,CT-120 and CT123) and a negative control antibody (CT-P6) wasadministered to mice by intra-abdominal injection in an amount of 10mg/kg of mice 24 hours before virus infection and 48 hours after virusinfection. The experimental results are shown in FIG. 6. As shown inFIG. 6, when the CT-104 or the CT-120 was injected into mice 24 hoursbefore infection with 10×LD₅₀ of H5N1 subtype virus(A/Vietnam/1203/2004), all the mice survived, but when mice were treatedwith the CT-123, 20% of the mice died after 12 days. In the case of thenegative control antibody (CT-P6), the mice injected with the controlantibody all died after 7 days (FIG. 6A). When the antibodies wereinjected 2 days after virus infection in order to examine thetherapeutic effects of the antibodies, the mice injected with the CT-104and the CT-120 all survived up to day 14, the last day of theobservation period, but the mice injected with the negative controlantibody (CT-P6) or the CT-123 all died (FIG. 6B).

When the antibodies were injected 24 hours before infection with thepandemic H1N1 subtype virus (A/California/07/2009) in order to examinethe preventive effects of the antibodies, the mice injected with theCT-120 and the CT-123 all survived up to day 14, the last day of theobservation period, and 80% of the mice injected with the CT-104survived, but the mice injected with the negative control antibody(CT-P6) all died (FIG. 6C).

In addition, the mice administered with the CT-104 or the CT-123 24hours before infection with the seasonal H1N1 subtype virus (A/PuertoRico/8/1934) all mice survived for the observation period, and the miceadministered with the CT-120 showed a survival rate of 80%, but the miceinjected with the negative control antibody (CT-P6 antibody) all died(FIG. 6D).

Example 6-2: Ferret Experiment

To investigate the curative virtues, the selected CT120 was tested onferret animal model, which shows similar sensitivities and symptoms tothat of human against influenza virus.

Each test group was composed of 9 ferrets except negative control groupincluding additional 4 ferrets to measure the initial concentration ofviral infection. Ferrets were intranasally or intratracheally inoculatedwith 1 ml (1×10⁶ EID₅₀/ml) of the influenza virus [A/California/04/09(H1N1)] after acclimatization. CT120 was intravenously injected once at24 hr after viral inoculation: test group 1 was injected with 15 mg/kgof CT120; test group 2 was injected with 30 mg/kg of CT120. For testgroup 3, 30 mg/kg of CT120 was injected every 24 hr for 3 days. Fornegative control group, 30 mg/kg of CT-P6 antibody was intravenouslyinjected once at 24 hr after viral inoculation.

Each nasal wash was collected from ferrets of each test group at 1, 3,5, and 8 days after viral inoculation and the viral concentrations incollected samples were measured using fertile eggs. 3 ferrets of eachtest group were sacrificed at 1, 3, 5, and 8 days after viralinoculation and the viral concentrations in removed lung tissues weremeasured using fertile eggs.

Each lung tissue was ground using homogenizer in PBS includingantibiotics (1 ml for each 1 g of lung tissues) and then supernatant wasremoved following centrifugation.

Each nasal wash was collected with 1 ml of PBS including antibiotics andthen supernatant was removed following centrifugation to measure theviral concentration. Supernatants of either lung tissue homogenate ornasal wash was ten-fold serially diluted with PBS including antibioticsand then 10-13 day old fertile eggs were inoculated with the dilutedsupernatant. The mixtures of allantoic fluid (50 μl) from 48hours-incubated fertile eggs and the same volume of 0.5% red blood cells(turkey) were incubated for 30 minutes and then virus was titrated byagglutination of blood.

Although viral titer in nasal wash remained high (>log 10 4 EID₀/ml)until 5 day post inoculation and then decreased in control group, viraltiter was significantly decreased in CT120-injected group and no viruswas detected at 8 day post-inoculation (FIG. 7). Thus, more rapid viralclearance was observed in CT120-treated group than in control group.Especially, virus was more significantly suppressed when the antibodywas injected daily for the initial 3 days in test group 3.

Viral titer in lung tissues remained high (>log 4.5 EID₅₀/me) until 5day post challenge and then decreased in control group, whereas viraltiter was markedly decreased in CT120-injected group. No virus wasdetected at 8 day post-challenge (FIG. 8). Especially, ferret experimentshowed that virus is more significantly suppressed in test group 2 and3. These results demonstrate that 30 mg/kg of CT120 more effectivelysuppress viral proliferation than 15 mg/kg dosage.

The invention claimed is:
 1. An anti-influenza A virus monoclonalantibody selected from the group consisting of the following monoclonalantibodies: (a) a monoclonal antibody comprising a light chaincomprising a CDR1 region comprising a sequence of SEQ ID NO: 1, a CDR2region comprising a sequence of SEQ ID NO: 2 and a CDR3 regioncomprising a sequence of SEQ ID NO: 3, and a heavy chain comprising aCDR1 region comprising a sequence of SEQ ID NO: 4, a CDR2 regioncomprising a sequence of SEQ ID NO: 5 and a CDR3 region comprising asequence of SEQ ID NO: 6; (b) a monoclonal antibody comprising a lightchain comprising a CDR1 region comprising a sequence of SEQ ID NO: 7, aCDR2 region comprising a sequence of SEQ ID NO: 2 and a CDR3 regioncomprising a sequence of SEQ ID NO: 8, and a heavy chain comprising aCDR1 region comprising a sequence of SEQ ID NO: 9, a CDR2 regioncomprising a sequence of SEQ ID NO: 10 and a CDR3 region comprising asequence of SEQ ID NO: 11; and (c) a monoclonal antibody comprising alight chain comprising a CDR1 region comprising a sequence of SEQ ID NO:12, a CDR2 region comprising a sequence of SEQ ID NO: 2 and a CDR3region comprising a sequence of SEQ ID NO: 13, and a heavy chaincomprising a CDR1 region comprising a sequence of SEQ ID NO: 14, a CDR2region comprising a sequence of SEQ ID NO: 15 and a CDR3 regioncomprising a sequence of SEQ ID NO: 16, wherein the monoclonal antibodyis produced by a non-human mammalian cell culture.
 2. The anti-influenzaA virus monoclonal antibody of claim 1, wherein the monoclonal antibodycomprises a light chain comprising a polypeptide sequence of SEQ ID NO:36, and a heavy chain comprising a polypeptide sequence of SEQ ID NO:37.
 3. The anti-influenza A virus monoclonal antibody of claim 1,wherein the monoclonal antibody comprises a light chain comprising asequence of SEQ ID NO: 40, and a heavy chain comprising a sequence ofSEQ ID NO:
 41. 4. The anti-influenza A virus monoclonal antibody ofclaim 1, wherein the monoclonal antibody comprises a light chaincomprising a polypeptide sequence of SEQ ID NO: 44, and a heavy chaincomprising a polypeptide sequence of SEQ ID NO:
 45. 5. Theanti-influenza A virus monoclonal antibody of claim 1, wherein thenon-human mammalian cell culture is CHO cells, COS cells or BHK cells.6. An anti-influenza A virus monoclonal antibody selected from the groupconsisting of the following monoclonal antibodies: (a) a monoclonalantibody comprising a light chain comprising a CDR1 region comprising apolypeptide encoded by a polynucleotide sequence set forth in SEQ ID NO:17, a CDR2 region comprising a polypeptide encoded by a polynucleotidesequence set forth in SEQ ID NO: 18 and a CDR3 region comprising apolypeptide encoded by a polynucleotide sequence set forth in SEQ ID NO:19, and a heavy chain comprising a CDR1 region comprising a polypeptideencoded by a polynucleotide sequence set forth in SEQ ID NO: 20, a CDR2region comprising a polypeptide encoded by a polynucleotide sequence setforth in SEQ ID NO: 21 and a CDR3 region comprising a polypeptideencoded by a polynucleotide sequence set forth in SEQ ID NO: 22; (b) amonoclonal antibody comprising a light chain comprising a CDR1 regioncomprising a polypeptide encoded by a polynucleotide sequence set forthin SEQ ID NO: 23, a CDR2 region comprising a polypeptide encoded by apolynucleotide sequence set forth in SEQ ID NO: 18 and a CDR3 regioncomprising a polypeptide encoded by a polynucleotide sequence set forthin SEQ ID NO: 24, and a heavy chain comprising a CDR1 region comprisinga polypeptide encoded by a polynucleotide sequence set forth in SEQ IDNO: 25, a CDR2 region comprising a polypeptide encoded by apolynucleotide sequence set forth in SEQ ID NO: 26 and a CDR3 regioncomprising a polypeptide encoded by a polynucleotide sequence set forthin SEQ ID NO: 27; or (c) a monoclonal antibody comprising a light chaincomprising a CDR1 region comprising a polypeptide encoded by apolynucleotide sequence set forth in SEQ ID NO: 28, a CDR2 regioncomprising a polypeptide encoded by a polynucleotide sequence set forthin SEQ ID NO: 29 and a CDR3 region comprising a polypeptide encoded by apolynucleotide sequence set forth in SEQ ID NO: 30, and a heavy chaincomprising a CDR1 region comprising a polypeptide encoded by apolynucleotide sequence set forth in SEQ ID NO: 31, a CDR2 regioncomprising a polypeptide encoded by a polynucleotide sequence set forthin SEQ ID NO: 32 and a CDR3 region comprising a polypeptide encoded by apolynucleotide sequence set forth in SEQ ID NO:
 33. 7. Theanti-influenza A virus monoclonal antibody of claim 6, wherein thebinding molecule comprises a light chain comprising a polypeptidesequence of SEQ ID NO: 34, and a heavy chain comprising a polypeptidesequence of SEQ ID NO:
 35. 8. The anti-influenza A virus monoclonalantibody of claim 6, wherein the binding molecule comprises a lightchain comprising a sequence of SEQ ID NO: 38, and a heavy chaincomprising a sequence of SEQ ID NO:
 39. 9. The anti-influenza A virusmonoclonal antibody of claim 6, wherein the binding molecule comprises alight chain comprising a polypeptide sequence of SEQ ID NO: 42, and aheavy chain comprising a polypeptide sequence of SEQ ID NO:
 43. 10. Theanti-influenza A virus monoclonal antibody of claim 6, wherein themonoclonal antibody is produced by a non-human mammalian cell culture.11. The anti-influenza A virus monoclonal antibody of claim 10, whereinthe non-human mammalian cell culture is CHO cells, COS cells or BHKcells.
 12. A composition comprising the anti-influenza A virusmonoclonal antibody of claim
 1. 13. A composition for preventing andtreating a disease caused by influenza A virus, the compositioncomprising the anti-influenza A virus monoclonal antibody of claim 1.14. A composition for diagnosis of influenza A virus, the compositioncomprising a conjugate comprising a tag linked to the anti-influenza Avirus monoclonal antibody of claim
 1. 15. The composition of claim 14,wherein the tag is any one selected from the group consisting ofenzymes, luciferases, radioactive isotopes, and toxin.
 16. A kit fordiagnosis of influenza A virus, comprising: i) the anti-influenza Avirus monoclonal antibody of claim 1; and ii) a container.
 17. The kitof claim 16, wherein the influenza A virus is selected from the groupconsisting of H 1, H2, and H5 subtypes.
 18. A composition comprising theanti-influenza A virus monoclonal antibody of claim
 6. 19. A compositionfor preventing and treating a disease caused by influenza A virus, thecomposition comprising the anti-influenza A virus monoclonal antibody ofclaim
 6. 20. A composition for diagnosis of influenza A virus, thecomposition comprising a conjugate comprising a tag linked to theanti-influenza A virus monoclonal antibody of claim
 6. 21. Thecomposition of claim 20, wherein the tag is any one selected from thegroup consisting of enzymes, luciferases, radioactive isotopes, andtoxin.
 22. A kit for diagnosis of influenza A virus, comprising: i) theanti-influenza A virus monoclonal antibody of claim 6; and ii) acontainer.
 23. The kit of claim 22, wherein the influenza A virus isselected from the group consisting of H 1, H2, and H5 subtypes.